Thyroid-epididymal relationship. I. Influence of hypothyroidism on epididymal lipids

The influence of thyroidectomy on the lipid composition of caput and cauda epididymides have been studied. The analysis was conducted in epididymal tissues free from fluids and sperm. A general tendency towards accumulation of epididymal lipids was observed in hypothyroid rats. Hypothyroidism also brought about a differential regional response, which may be age-related. The existence of a relationship between triacylglycerols, phospholipids and diacylglycerols has been suggested. Since immediate thyroxine replacement to thyroidectomised rats maintained epididymal lipids at control levels, it is concluded that hypothyroidism has a definite influence on the epididymal lipid composition.     

Effect of corticosterone on epididymal lipids in pubertal rat

Serum corticosterone excess was induced by the administration of corticosterone acetate to adrenal intact rats. Different lipid classes were studied in unwashed and washed (epididymal sperm and fluid free) caput and cauda epididymides. The unwashed caput epididymidis registered a significant decrease in total lipid, cholesterol and phospholipid while total glyceride glycerol and its fractions were not altered after corticosterone treatment. Among phospholipid fractions phosphatidyl inositol, choline and ethanolamine showed a significant decrease. Unlike the unwashed caput epididymidis, the washed caput region recorded a marked increase in total lipid, glyceride glycerol and its fractions. However, total lipid in the washed cauda region significantly increased and the increase was mainly due to triacyl glycerol. Though the phospholipid fractions phosphatidyl choline and ethanolamine showed an increase, the total phospholipid was not altered significantly. Serum testosterone and prolactin registered a significant decrease while gonadotropins were unaltered. On the withdrawal of corticosterone treatment, all the lipid classes turned to normalcy along with serum testosterone and prolactin. It is concluded that corticosterone excess favours lipid accumulation in the sperm free epididymal tissue and its influence on epididymis is region specific and reversible.     

Effects of castration on thiol status in rat spermatozoa and epididymal fluid

Mammalian spermatozoa gain their fertilizing ability as they mature in the epididymis, a process which is accompanied by oxidation of sperm protein thiols. Since sperm maturation is dependent upon normal androgenic support to the epididymis, the present work was designed to study the effects of castration on thiol status. Spermatozoa and epididymal fluid were isolated from the epididymides of male rats 5 days after castration or after 11 daily injections of the antiandrogen, cyproterone acetate. Spermatozoa and epididymal fluid were labeled with the fluorescent thiol labeling agent monobromobimane. Intact spermatozoa were evaluated by fluorescence microscopy, protein thiols were analyzed by electrophoresis, and fertilizing ability was examined after insemination of sperm suspension into the uterine horns of immature superovulated female rats. We found that both treatments resulted in an increase in cauda sperm thiols as shown by increased fluorescence in the intact spermatozoa. Protamines and nonbasic proteins were found to have increased levels of reactive thiols. The protein profiles of epididymal fluid from castrated rats were different from those of the controls, and the fluorescence patterns corresponded to the protein profiles. Our results indicate that testosterone withdrawal leads to inhibition of sperm thiol oxidation. Mol. Reprod. Dev. 47:295–301, 1997. © 1997 Wiley-Liss, Inc.     

Rat sperm enzymes during epididymal transit

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Steroidogenic characteristics of in vitro cultured epididymal epithelial cells of the rat

The paper presents the steroidogenic features of cultured epithelial cells of rat epididymis and their ability to synthesize steroid hormones. The cytoplasm of epididymal epithelial cells accumulated lipid droplets and contained active enzymes of steroidogenesis. Numerous mitochondria with lamellar cristae occurred near lipid droplets. Frequently, mitochondria formed a direct contact with lipid droplets and smooth endoplasmic reticulum. The hormone assay showed that the epididymal epithelial cells cultured without dihydrotestosterone synthesized and released the following steroids: dehydroepiandrosterone (DHEA), testosterone (T) and 17beta-estradiol (E). The levels of DHEA and T were very low. The concentration of E detected in media of cultured epididymal epithelial cells exceeded many times the concentration of E in control media. The cytoplasmic presence of organelles and enzymes that participated in the steroid synthesis indicated their similarity to steroidogenic cells. Epididymal epithelial cells were capable of moderate in vitro synthesis of androgens. It cannot be excluded that steroidogenesis in the cultured epididymal epithelial cells is maintained to sustain 17beta-estradiol synthesis pathways.     

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.     

Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit

Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda.     

Glycoproteins of ram sperm plasma membrane. Relationship of protein having affinity for Con A to epididymal maturation

Con A Receptors from the sperm plasma membrane were quantitated (using ³H acetyl-Con A) along the epididymal duct; they diminished in the second part of the epididymis as compared to the epididymal head. Glycoproteins having affinity for Con A were partially characterized: washed spermatozoa from rete testis (= testicular spermatozoa), middle corpus and distal cauda epididymis were labelled (¹²⁵I Na). Proteins of their plasma membrane were extracted (Triton ×100, 0.1% and chromatography affinity): differences appeared in ACA44 profiles from ¹²⁵I Con A Glycoprotein extractions between testicular spermatozoa (2 major peaks Kav= 0.41 and 0.52) and epididymal spermatozoa (3 major peaks Kav= 0.33–0.34, 0.41 and 0.52 and additional minor peaks between 0.66 and 1.00). The peak Kav= 0.41 diminished considerably on epididymal spermatozoa as compared to testicular spermatozoa.     

Maturation-dependent goat epididymal sperm autoagglutination and its inhibition by a glycoprotein factor.

The maturing goat epididymal spermatozoa were isolated from different segments of epididymis and these cells dispersed in a modified Ringer's solution, were incubated at 37 degrees C for 60 min to evaluate their autoagglutination efficacy. Distal corpus-epididymal spermatozoa specifically showed high order of head-to-head autoagglutination property whereas all other sperm cells did not show any detectable aggregation. The goat epididymal plasma has been shown to possess an anti-agglutinin that markedly inhibits sperm agglutination phenomenon and also dissociates the cells from the sperm clusters. Epididymal plasma is the most potent source of the anti-agglutinin which is a heat-stable specific glycoprotein. Like the autoagglutination phenomenon, the initiation of sperm forward progression also starts in the distal-corpus epididymis. The temporal correlation of these two events suggests that sperm autoagglutination may be a prerequisite for the induction of flagellar motility during the epididymal maturity of male gametes.     

Rat epididymal epithelial cells and 17beta-estradiol synthesis under hCG stimulation in vitro

Epithelial cells of human and animal epididymis display features of steroidogenic cells. Rat epididymal epithelial cells in vitro produce androgens which are converted to 17beta-estradiol, and released into the medium. The regulation of the epididymal steroidogenesis is not fully understood but it could be expected that it remains under LH influence. In previous study we observed that the morphology of rat epididymal epithelial cells in vitro was affected by hCG and the increase of amount of lipid droplets, glycogen and PAS-positive substances was observed. The present studies show the organelles which take part in synthesis of steroids in rat epididymal epithelial cells in vitro and the effect of hCG on E2 synthesis. The cells were cultured in the medium with/without DHT and without DHT in supplementation with hCG. After hCG stimulation the amount of an active mitochondria were increased when compared to the amount of mitochondria in the epididymal epithelial cells cultured without DHT. Ultrastructure of the cells was similar to the cells cultured with DHT, while the cytoplasm of the cells cultured without DHT was disorganized. The synthesis of 17beta-estradiol was stimulated by hCG, that exerted its effect through LH/hCG receptors, localized in the epididymal epithelial cells.     

Effect of epididymal isolation on the protein component of bull eididymal tissue.

Four fractions of protein from intact and 'isolated' bull epididymides were extracted. The total extractable protein was increased by isolation mainly due to increases in the lipoprotein and DNA-protein fractions. Changes of lipoprotein may be associated with the increase of lipid which also occurs in isolated tissue. This may reflect the increase of pentose cycle activity and lipogenesis due to the absence of spermatozoa in the epididymal tubules. Changes of DNA-protein may indicate a change of protein metabolism. Electrophoresis of protein fractions showed that individual proteins may be changed by the zonal origin of the epididymal tissue and the presence or absence of spermatozoa in the epididymal tubules.     

Cryopreservation of epididymal stallion sperm

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.     

Nonpathological extracellular amyloid is present during normal epididymal sperm maturation

Amyloids are aggregated proteins characterized by a specific cross-β-sheet structure and are typically associated with neurodegenerative diseases including Alzheimer's disease. Recently, however, several nonpathological amyloids have been found in intracellular organelles of normal mammalian tissues suggesting that amyloid may also carry out biological functions. We previously have shown that the epididymal cystatin CRES (cystatin-related epididymal spermatogenic), cst8, a reproductive-specific member of the cystatin superfamily of cysteine protease inhibitors, forms amyloid in vitro suggesting that CRES amyloid may also form in vivo within the epididymal lumen. Here we show that amyloid structures containing CRES are a component of the normal mouse epididymal lumen without any apparent cytotoxic effects on spermatozoa and that these structures change along the length of the tubule. These studies suggest the presence of a functional amyloid structure that may carry out roles in sperm maturation or maintenance of the luminal milieu and which itself may undergo maturational changes along the epididymis. In contrast to previous examples of functional amyloid which were intracellular, our studies now show that nonpathological/functional amyloid can also be extracellular. The presence of an extracellular and nonpathological amyloid in the epididymis suggests that similar amyloid structures may be present in other organ systems and may carry out distinctive tissue-specific functions.     

Changes in the lipid content of boar sperm plasma membranes during epididymal maturation

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm3. This fraction was enriched in acid and alkaline phosphatase, 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.     

Morphology and motility of spermatozoa from different regions of the epididymal duct in the domestic cat

Spermatozoa undergo important maturational changes as they pass through the epididymal duct. Some domestic cats and many species of wild felids have high proportions of abnormal spermatozoa in their ejaculates. The epididymis has been shown to be able to remove certain abnormal sperm forms in some species while other sperm abnormalities originate in the epididymis. So far, it has not been shown how the epididymis affects sperm morphology in the domestic cat. Therefore, motility and sperm morphology were studied in spermatozoa from the efferent ducts and from the 6 regions of the epididymal duct. There were significant decreases in the proportions of spermatozoa with abnormalities of the sperm head, acrosomal defects, acrosomal abnormalities and in the proportion of midpiece abnormalities. In contrast, there was a small but significant increase in the proportion of spermatozoa with abnormalities of the tail. Spermatozoa acquired the capacity for motility in Region 4, where the cytoplasmic droplet also moved from a proximal to a distal position, indicating that important maturational changes take place in this region. The results of this study demonstrate that the proportions of sperm abnormalities originating in the testes decrease during epididymal transport, while some sperm tail abnormalities may actually originate in the epididymis.     

Creatine phosphokinase in domestic cat epididymal spermatozoa

Mammalian spermatozoa that have not completed final testicular sperm maturation have residual cytoplasm and increased creatine phosphokinase (CK) content. This study determined: (1) if CK could be detected by immunostaining cat spermatozoa from the caput, corpus, and cauda epididymis, (2) fluctuations in the proportions of spermatozoa with mature or immature CK-staining patterns during epididymal sperm transit, and (3) how well sperm maturity (as determined by a CK marker) correlated with testicular or epididymal dysfunctions associated with morphological sperm abnormalities. One epididymis was collected from each of 37 cats after orchiectomy and processed immediately to allow sperm morphology evaluations on a 'regional' basis. Sperm released from the contralateral epididymis were evaluated for motility, sperm membrane integrity, and immunostaining with CK-B antibodies. Proportions of spermatozoa with malformed or detached heads, proximal droplets and acrosomal or midpiece abnormalities decreased (P < 0.05) from the caput to the cauda epididymis. In contrast, proportions of spermatozoa that were motile, membrane-intact or with flagellar abnormalities or distal droplets increased (P < 0.05) from the caput to cauda region. Percentages of spermatozoa with an immature CK-staining pattern also decreased (P < 0.05) with epididymal transit (which differs from that reported for the human and stallion). There was no correlation (P > 0.05) between sperm morphology and the CK-staining patterns. In summary, the results reveal that some specific sperm malformations in the domestic cat are of testicular origin, whereas others develop during epididymal transit.     

Use of titanium dioxide to find phosphopeptide and total protein changes during epididymal sperm maturation

Although the overall performance of modern mass spectrometers has increased, proteomic analysis of complex samples still requires prefractionation either at the protein or peptide level to allow for in-depth analysis of normal cellular function. Here, we report a novel way to identify protein changes occurring during sperm development through the epididymis. Phosphopeptides were first enriched from either the rat caput or caudal regions of the epididymides using TiO(2), and the profiles then quantitatively compared. We show that 77 TiO(2)-enriched peptides become significantly modified in the epididymis, equating to 53 proteins. Through the use of immunoblot analysis, we confirmed that three proteins, ornithine-decarboxylase antizyme 3, heat-shock protein 90α, and testis-lipid binding protein, undergo major protein loss during epididymal passage. Many other proteins, including t-complex protein 10 and Spata18 show testis unique expression, appear to undergo phosphorylation during this same time frame. These data provide mechanistic insight into the means by which spermatozoa acquire functionality during epididymal transit.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.